Abstract Bacterial infections during neonatal phase cause high rates of morbidity and mortality, and in developing countries are responsible for 26% of deaths. Environmental factors present during pregnancy are known to impact life-threatening infections in newborns, including Staph. aureus infections, although the mechanisms are unclear. Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental pollutant, which acts through the cytosolic aryl hydrocarbon receptor (AhR). While AhR has been well characterized for its role in regulating toxicity mediated by TCDD, recently, AhR activation was shown to regulate T cell differentiation into T regs or Th17 cells. We have generated exciting preliminary data indicating that AhR activation by TCDD suppresses T cell response to Staphylococcal enterotoxin B (SEB) and that this is mediated by epigenetic pathways including dysregulation in microRNA (miR) expression, DNA methylation, and histone modifications in activated T cells. More importantly, our studies have suggested that TCDD may exert transgenerational epigenetic effects on T cells. Based on the importance of Staph infections discussed above, we will use SEB as an antigen to test the central hypothesis that AhR activation of V?8+ T cells by TCDD, plays a crucial role in reducing pro-inflammatory Th1/Th17 cells as well as increasing anti-inflammatory Tregs and its subsets by modulating miR expression, and that this may depend on DNA methylation, histone modifications and chromatin remodeling that could be transmitted transgenerationally. Inasmuch as, SEB can activate V?8+ T cells which constitute ~30% of peripheral T cells, our studies are aimed at determining whether TCDD-induced changes persist in F0, F1, F2, and F3 generations following maternal exposure during pregnancy to TCDD or maternal/paternal exposure prior to mating (F0). In Aim 1, we will determine the transgenerational effects of TCDD on SEB-induced CD4+ T cell differentiation. We will test the effect of TCDD on the TCR clonality and diversity of the V?8+ CD4+ T cell response (Th1, 2, 17, Tregs) to SEB. In Aim 2, we will study the role of specific miRs in CD4+ T cell differentiation in F0-F3 generations. Furthermore, transfection of T cells with specific miR mimics or antagomirs will be performed to reverse T cell differentiation induced by TCDD and determine whether the effects persist across generations. In Aim 3, we will determine the role of genome-wide and locus-specific DNA methylation on CpG sites on promoters of specific miR that regulate differential expression of Th/Treg response to SEB across the generations. Aim 4 will elucidate the permissive and repressive histone modification and chromatin accessibility in TCDD-mediated transgenerational dysregulation of miR involved in CD4+ T cell differentiation. Lastly, whether these changes are imprinted through male or female germline will be assessed. The proposed studies are highly significant in that novel epigenetic pathways of TCDD-mediated immunotoxicity across generations will be identified. Also, understanding how AhR ligands mediate differential effects through epigenetic pathways would lead to development of innovative preventive and therapeutic modalities.